Anti inflammatory effects of inhibitors of the tyrosine kinase signaling cascade in animal models of asthma

ANTI-INFLAMMATORY EFFECTS OF INHIBITORS OF THE TYROSINE KINASE SIGNALING CASCADE IN ANIMAL MODELS OF ASTHMA Duan Wei (B.Med.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENTS No project is a solo venture Numerous people, across these years, are a part of this project To name just a few: • My supervisor, Professor Fred Wong, who has picked me up, dusted me off, and set me back on track of this project more than once His attitude and discipline are just some of the lessons that I believe I will carry with me for the rest of my life • Professor Chua Kaw Yan and Professor Chang Chan Fong, who kindly provided me help and shared with me their experiences • My good friends, Yanan, Olivia, Rebecca, Yihan, Constance & Chee Kiong, Cheng Ann & Christine, Uncle Richard and Aunt Tsu Yeow, and Hock Chuan for their friendship sharing • Thanks also go to National University of Singapore for providing me years scholarship and the staff & postgraduate students of the Department of Pharmacology • Finally, my parents, whose endless love and supports surround me all the time Duan Wei March 2004 ii CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF FIGURES viii LIST OF TABLE xii LIST OF ABBREVIATIONS xiii LIST OF PUBLICATIONS AND CONFERENCE PAPERS xv SUMMARY xvii INTRODUCTION 1.1 Asthma 1.1.1 Pathophysiology of asthma 1.1.1.1 Mast cells and acute bronchoconstriction 1.1.1.2 Eosinophilia 1.1.1.3 T lymphocytes and Th2 cytokines 1.1.1.4 B cells and immunoglobulins 15 1.1.1.5 Airway mucus hypersecretion 17 1.1.1.6 Airway hyperresponsiveness (AHR) 18 1.1.2 Therapeutic targets of asthma 20 1.1.2.1 Current therapy for asthma 21 1.1.2.2 Novel therapeutic targets for asthma 22 1.2 Tyrosine kinase signaling cascade 1.2.1 Protein tyrosine kinases (PTKs) 25 25 iii 1.2.1.1 Role of PTK signaling cascade in allergic inflammation 1.2.2 Mitogen-activated protein kinase (MAPK) cascades 28 29 1.2.2.1 MAPK 29 1.2.2.2 Role of MAPK cascade in allergic inflammation 31 1.2.3 Phosphoinositide kinase (PI3K) 33 1.2.3.1 PI3K cascade 33 1.2.3.2 Role of PI3K cascade in allergic inflammation 37 1.3 Inhibitors of the tyrosine kinase signaling cascade 38 1.3.1 Protein tyrosine kinase inhibitors 38 1.3.2 MAPK kinase inhibitors 40 1.3.3 PI3K inhibitors 43 1.4 Animal models of asthma 45 RATIONALE AND OBJECTIVES 50 METHODS AND MATERIALS 52 3.1 Animal sensitization and challenge 53 3.1.1 Guinea pigs 53 3.1.2 Mice 53 3.2 Measurement of bronchoconstriction and AHR 53 3.2.1 Measurement of bronchoconstriction in guinea pigs 55 3.2.2 Measurement of AHR in mice 55 3.3 Bronchoalveolar lavage (BAL) fluid collection and cytospin 57 3.3.1 Collection of BAL fluid from guinea pigs 57 3.3.2 Collection of BAL fluid from mice 58 3.4 ELISA 61 iv 3.4.1 Cytokines and chemokine measurement in BAL fluid 61 3.4.2 Immunoglobulin measurement in serum 62 3.5 Eosinophil peroxidase (EPO) measurement 62 3.6 Histology 63 3.7 Immunohistochemistry 65 3.8 Western blotting 65 3.8.1 Analysis for phospho-tyrosine residues 65 3.8.2 Analysis for phospho-ERK and phospho-Akt 67 3.9 Data analysis 67 3.10 Materials 67 Anti-inflammatory effects of genistein, a tyrosine kinase inhibitor, in a guinea pig model of asthma 70 4.1 Results 71 4.1.1 Effects of genistein on OVA-induced acute bronchoconstriction 71 4.1.2 Effects of genistein on OVA-induced airway inflammation 76 4.1.3 Effects of genistein on AHR 81 4.1.4 Phosphotyrosine immunoblot analysis 86 4.2 Discussion 86 4.2.1 Genistein attenuates OVA-induced acute bronchoconstriction 86 4.2.2 Genistein attenuates OVA-induced airway inflammation 88 4.2.3 Genistein attenuates OVA-induced AHR 89 Anti-inflammatory effects of a MAPK kinase inhibitor, U0126, in a mouse asthma model 91 v 5.1 Results 92 5.1.1 Effects of U0126 on OVA-induced eosinophil recruitment in BAL fluid 92 5.1.2 Effects of U0126 on OVA-induced eosinophil infiltration and mucus production 92 5.1.3 Effects of U0126 on cytokine levels in BAL fluid 97 5.1.4 Effects of U0126 on serum immunoglobulin levels 97 5.1.5 Effects of U0126 on the expression of VCAM-1 in lung tissues 103 5.1.6 Effects of U0126 on AHR 103 5.1.7 Phospho-ERK immunoblot analysis 108 5.2 Discussion 5.2.1 U0126 reduces cytokine levels in BAL fluid 108 5.2.2 U0126 attenuates OVA-induced airway and lung tissue eosinophilia 114 5.2.3 U0126 attenuates OVA-induced mucus production 115 5.2.4 U0126 reduces serum immunoglobulin levels 116 5.2.5 U0126 attenuates OVA-induced AHR 116 Anti-inflammatory effects of LY294002, a specific PI3K inhibitor, on a mouse model of asthma 118 6.1 Results 119 6.1.1 Effects of LY294002 on OVA-induced eosinophil recruitment in BAL fluid 119 6.1.2 Effects of LY294002 on OVA-induced eosinophil infiltration and mucus production 119 vi 6.1.3 Effects of LY294002 on cytokine levels in BAL fluid 123 6.1.4 Effects of LY294002 on expression of VCAM-1 in lung tissue 123 6.1.5 Effects of LY294002 on AHR 123 6.1.6 Phospho-Akt immunoblot analysis 129 6.2 Discussion 6.2.1 LY294002 reduces cytokine levels in BAL fluid 129 6.2.2 LY294002 attenuates OVA-induced airway and lung tissue eosinophilia 131 6.2.3 LY294002 attenuates OVA-induced mucus production 132 6.2.4 LY294002 attenuates OVA-induced AHR 133 Conclusion 134 References 137 vii LIST OF FIGURES Figure Title Page 1.1 Schematic diagram of pathogenesis of asthma 1.2 Schematic diagram of protein tyrosine kinase signaling cascade 27 1.3 Schematic diagram of MAPK signaling cascade 30 1.4 Schematic diagram of PI3K signaling cascade 35 1.5 Structures of PTK inhibitors 39 1.6 Structures of MAPK kinase (MEK) inhibitors 42 1.7 Structures of PI3K inhibitors 44 3.1 Aerosol delivery system 53 3.2 Buxco system 54 3.3 Penh 56 3.4 BAL fluid cells (guinea pig) 59 3.5 BAL fluid cells (mouse) 60 4.1 Representative tracings showing the changes in box pressure waveform 4.2 Inhalation of OVA-induced acute bronchoconstriction in guinea pigs 4.3 74 Effects of genistein or daidzein on OVA-induced bronchoconstriction in guinea pigs 4.4 72 75 Effects of genistein on histamine-induced acute bronchoconstriction 77 viii 4.5 Effects of genistein on methacholine-induced acute bronchoconstriction 4.6 OVA-induced BAL fluid eosinophilia in sensitized guinea pigs 4.7 85 Effects of genistein on OVA-induced AHR to inhaled methacholine in sensitized guinea pigs 4.12 84 OVA-induced AHR to inhaled methacholine in sensitized guinea pigs 4.11 83 Histologic examination of inflammatory cell infiltration into the bronchioles in guinea pigs 4.10 82 Histologic examination of inflammatory cell infiltration into the trachea in guinea pigs 4.9E-H 80 Effects of genistein on EPO activity in cell-free BAL fluid in guinea pigs 4.9A-D 79 Effects of genistein or daidzein on OVA-induced BAL fluid eosinophilia in guinea pigs 4.8 78 86 Effects of genistein on EGF-induced protein tyrosine phosphorylation in guinea pig lung fragments 88 5.1 OVA-induced cell infiltration in sensitized mice 94 5.2 Effects of U0126 on BAL fluid cell infiltration 95 5.3 Effects of U0126 on BAL fluid cell infiltration from sensitized mice challenged with saline 5.4 96 Effects of U0124 on BAL fluid cell infiltration from sensitized mice challenge with OVA 5.5A-D, I 97 Effects of U0126 on lung tissue eosinophilia 99 ix 5.5E-H, J Effects of U0126 on mucus production 100 5.6 Effects of U0126 on cytokine levels in BAL fluid 101 5.7 Effects of U0126 on the eotaxin level in BAL fluid 102 5.8 Effects of U0126 on the IFNγ level in BAL fluid 103 5.9 Effects of U0126 on serum total and OVA-specific IgE production 105 5.10 Effects of U0126 on serum IgG1 and IgG2a production 106 5.11 Effects of U0126 on the expression of VCAM-1 in lung tissue 107 5.12 OVA-induced AHR in sensitized mice 108 5.13 Effects of U0126 on OVA-induced AHR 110 5.14 Effects of U0126 on methacholine-induced AHR in sensitized mice challenged with saline aerosol 5.15 Effects of U0126 on OVA-induced ERK phosphorylation in sensitized mouse lung tissues 5.16 111 112 Effects of U0126 on OVA-induced ERK expression in sensitized mouse lung tissues 113 6.1 Effects of LY294002 on BAL fluid cell infiltration 121 6.2A-D, I Effects of LY294002 on lung tissue eosinophilia 122 6.2E-H, J Effects of LY294002 on mucus production 123 6.3 Effects of LY294002 on cytokine levels in BAL fluid 125 6.4 Effects of LY294002 on the eotaxin level in BAL fluid 126 6.5 Effects of LY294002 on the IFNγ level in BAL fluid 127 6.6 Effects of LY294002 on the expression of VCAM-1 in lung tissue 128 x 6.7 Effects of LY294002 on OVA-induced AHR to inhaled methacholine in sensitized mice 6.8 129 Effects of LY294002 on OVA-induced Akt phosphorylation in sensitized mouse lung tissues 131 xi LIST OF TABLE Table Title Page I Action of IL-13 on haematopoietic and nonhematopoietic 14 cells xii LIST OF ABBREVIATIONS AP APC BAL BCA BCIP BLNK BSA CCR CysLTs DAG DMSO ECP EGF EGFR EPO ERK FcεRI FcγR FITC GAB2 GM-CSF GPCR ICAM-1 Ig IL IP3 IRS1/2 ITAMs JAK/STAT LAT MAPK MAPKK MBP MCP MHC MIP-1α NBT OVA PAF PBS PDGF PDK1 PI3K PKB PKC PLC PMSF alkaline phosphatase antigen-presenting cells bronchoalveolar lavage bicinchoninic acid 5-bromo-4-chloro-3-indoyl-phosphate B-cell linker bovine serum albumin CC chemokine receptor cysteinyl-leukotrienes diacylglycerol dimethyl sulphoxide eosinophil cationic protein epidermal growth factor EGF receptor eosinophil peroxidase extracellular signal-regulated kinase Fc-epsilon receptor I Fc-gamma receptor fluorescein isothyiocyanate GRB2-associated binding protein granulocyte-macrophage colony-stimulating factor G-protein-coupled receptor intracellular adhesion molecule immunoglobulin interleukin inositol 1,4,5-triphosphate insulin receptor substrate-1/2 immunoreceptor tyrosine – based activation motifs Janus Kinase/signal transducer and activation of transcription linker for activation of T cells mitogen-activated protein kinase MAPK kinase major basic protein monocyte chemoattractant protein major histocompatibility complex macrophage inflammatory protein -1α nitroblue tetrazolium ovalbumin platelet-activating factor phosphate-buffered saline platelet derived growth factor phosphatidylinositol-dependent kinase phosphoinositide 3-kinase protein kinase B protein kinase C phospholipase C phenylmethanesulfonyl fluoride xiii PTEN PtdIns PtdIns 4-P PtdIns 3,4-P2 PtdIns 4,5-P2 PtdIns 3,4,5-P3 PTK PVDF RANTES RTK SDS SH SLP76 TCR VCAM-1 VLA-4 ZAP70 phosphatase and tensin homologue phosphatidylinositol phosphatidylinositol 4-phosphate phosphatidylinositol 3,4-biphosphate phosphatidylinositol 4,5-biphosphate phosphatidylinositol 3,4,5-triphosphate protein tyrosine kinase polyvinylidene difluoride regulated on activation, normal T cell expressed and secreted receptor tyrosine kinase sodium dodecyl sulfate src homology SH2-domain-containing leukocyte protein of 76 kDa T cell receptor vascular-cell adhesion molecule very late antigen-4 ξ-chain-associated protein kinase 70 kDa xiv LIST OF PUBLICATIONS AND CONFERENCE ABSTRACTS Publications Duan W, Kuo IC, Selvarajan S, Chua KY, Bay BH, and Wong WSF (2003) Antiinflammatory effects of genistein, a tyrosine kinase inhibitor, on a guinea pig model of asthma Am J Respir Crit Care Med 167(2): 18592 Chue SC, Seow CJ, Duan W, Yeo KSL, Koh AHM, and Wong WSF (2004) Inhibitor of p42/p44 mitogen-activated protein kinase (MAPK) kinase, but not p38 MAPK, attenuated antigen challenge of guinea pig airway in vitro Intl Immunopharma 4: 1089-1098 Yeo KSL, Zakaria SM, Duan W, Chan SY, Wong WSF Inhibitors of the tyrosine kinase signaling cascade attenuated acute atopic dermatitis in guinea pigs Submitted, 2003 Duan W, Chan HP, Leong KP, and Wong WSF (2004) Anti-inflammatory effects of a MAPK kinase inhibitor U0126 in a mouse asthma model J Immunol 172; 7053-7059 Duan W, Leong KP, Vlahos CJ, and Wong WSF Anti-inflammatory role of a phosphoinositide 3-OH kinase inhibitor LY294002 in a mouse asthma model accepted, 2005 Intl Immunopharmaco Conference Abstracts Duan W, Tsang F, Kuo IC, Chua KY & Wong WSF: Anti-inflammatory effects of tyrosine kinase inhibitors on guinea pig model of asthma The xv International Conference on Fundamental Sciences: Biological & Chemical Sciences, Singapore, P115, 2001 Wong WSF, Duan W, Tsang F, Sung CC, Shoon ML, Low KSY, Selvarajan S & Bay BH: Anti-inflammatory effects of inhibitors of the tyrosine kinase signaling cascade on a guinea pig model of asthma BioMedical Asia 2001 Singapore, ET-31, 2001 Duan W, Shoon ML, Low SYK, Selvarajan S, Bay BH & Wong WSF: Antiinflammatory effects of genistein, a protein tyrosine kinase inhibitor, on an in vivo guinea pig model of asthma Pharmacologist 44 (Suppl 1): Abst 134: 27, 2002 Yeo KSL, Duan W, Chan SY, Bay BH & Wong WSF: Quantitative analysis of atopic dermatitis in guinea pigs: Anti-allergic effects of inhibitors of the tyrosine kinase signaling cascade 6th NUS-NUH Annual Scientific Meeting, Singapore, 2002 Wong WSF, Yeo KSL, Duan W, Chan SY & Bay BH: Quantitative analysis of atopic dermatitis in guinea pigs: Anti-allergic effects of inhibitors of the tyrosine kinase signaling cascade World Congress on Immunopathology, Singapore Int’l J Immunorehab 4: Abst 8, 2002 Duan W, Kuo IC, Selvarajan S, Chua KY, Bay BH & Wong WSF: Antiinflammatory effects of genistein, a protein tyrosine kinase inhibitor, on an in vivo guinea pig model of asthma World Congress on Immunopathology, Singapore Int’l J Immunorehab 4: Abst 21, 2002 Duan W, Chan HP, Leong KP, and Wong WSF Anti-inflammatory effects of U0126, a specific MEK inhibitor, in a mouse model of asthma J World Allergy Org Supple 1, P:5, 2003 xvi Summary Protein tyrosine kinase signaling cascade plays a pivotal role in the activation of inflammatory cells Stimulation of nonreceptor tyrosine kinases is the earliest detectable signaling response upon immunoreceptor activation in mast cells, T and B cells, eosinophils, and macrophages It is believed that src-family kinases (Lyn and Lck) and Syk/ZAP-70 are responsible for the initial activation of these cells Subsequently, ERK signaling pathway is also activated leading to proliferation, differentiation, cytokine production and degranulation of inflammatory cells In addition, PI3K signaling pathway is activated upon T cell, B cell and mast cell activation There were no studies on the effects of the intervention of these signaling pathways in animal models of asthma The purpose of my thesis was to investigate the antiinflammatory effects of genistein, a broad-spectrum protein tyrosine kinase inhibitor; U0126, a specific MAPK/ERK kinase (MEK) inhibitor, and LY294002, a specific PI3K inhibitor in asthmatic animal models Guinea pigs were actively sensitized by intraperitoneal injections of ovalbumin Aerosolized ovalbumin induced acute bronchoconstriction in conscious animals in a dose-dependent manner Genistein (15 mg/kg given intraperitoneally) markedly (p